Over the past few decades, the discovery of a wide range of functional RNAs has revealed an expanding role for RNA in essential cellular processes such as gene expression and protein biosynthesis;however, the known chemical diversity available to RNA is still relatively limited. Traditionally, the discovery of chemically modified ribonucleotides in biological systems has been limited to a target based approach;either the class of RNA molecules or the nucleotide modification were known beforehand and studied in a limited context. Recently, a general screen has been reported for the discovery of small molecule-RNA conjugates. Unlike previous methods, this screen is not dependent on a specific small-molecule structure, on a certain class of RNA, or on a particular biological function of the conjugate. The identification of novel chemical modifications of RNA has the potential to improve our still relatively poor understanding of RNA biology in areas such as gene regulation or gene splicing, and, more provocatively, may identify entirely new roles for RNA in the cell, such as new modes of catalysis, signaling, or gene regulation. Previously, application of this approach led to the discovery of two novel 5'small molecule-RNA conjugates in two bacterial organisms. Here, the screen will be used to identify modified nucleotides present in yeast. Following identification, focus will be placed on characterizing the biological function of the modified nucleotides through development of sequencing methods for modified RNAs as well as attempting to link the modified RNA to a known biological process. The proposed research has the potential to provide a more complete understanding of the chemical diversity of cellular RNA in yeast, and possibly significantly expand our understanding of the biological, and perhaps even chemical, functions of RNA. PUBLIC HEALTH RELEVANCE: The known biological roles of cellular RNA have expanded considerably in the past few decades;however, the chemical diversity of RNA has remained relatively constant. This proposal seeks to identify novel modified RNA in yeast, an important eukaryotic model organism, and characterize their biological function. An increased understanding of the chemical diversity of RNA will potentially improve understanding of the biological role of RNA, and, eventually, lead to new therapeutic targets.